With recent rapid developments in molecular biology, considerable advance has been made in development of therapeutic agents for various cancers and other intractable diseases using recombinant DNA technologies. Also, since gene therapy was first attempted clinically in 1990, studies on delivery systems of therapeutic genetic materials have been carried out. Among clinical gene therapies, about 60% focus on treating cancers, and various vectors based on human or non-human viruses have been developed as vehicles to transfer therapeutic genes into target cells, in which adenovirus with many advantages as a gene transfer vehicle is attractive as a substitute for retroviruses.
For practical use of gene therapy in clinical fields, first of all, gene transfer vehicles capable of safely and effectively delivering therapeutic genes to targeted regions should be developed. The potential of recombinant adenovirus as a gene transfer vehicle was reported in 1984 by Graham, F. L. (Graham, F. L., EMBO J., 1:2917-2922, 1984). Since the first clinical trial of gene therapy in adenosine deaminase (ADA)-deficient patients in 1990, active research into gene therapy was performed until the mid-1990s, in which expression of a specific gene was induced in target cells using replication-defective recombinant adenoviral vectors constructed by inserting the specific gene thereinto. Such research was promoted by the fact that adenovirus has many benefits as a gene transfer vehicle, in terms of being excellent in transferring exogenous genes into a variety of cell types regardless of cell cycle state of target cells, easily producing high-titer virus, being capable of being lyophilized and thus structurally stable, and being easily formulated into pharmaceutical preparations (Yeh, P. et al., FASEB J., 11:615-622, 1997).
However, adenovirus has several significant problems in mediating gene transfer, including the relatively short-term expression of foreign genes and the induction of strong immune response to viral proteins and virus-infected cells. To overcome such problems, various efforts to modify structure of adenovirus have been made. For example, it was reported by S. Kochaneck in 1996 that gutless virus produced by removing all adenoviral coding sequences is advantageous in terms of being capable of carrying maximum 30 kb of foreign genes, as well as not producing viral proteins and thus not inducing immune response in host cells (Kochanek, S., Proc. Natl. Acad. Sci. 93:5731-5736, 1996). Korean Pat. Publication No. 1999-22941 discloses an adenovirus vector having no overlap with a suitable packaging cell line, in which adenovirus loses self-replication ability and is incapable of being encapsidated, thus avoiding interference with the host immune system.
Since recombinant adenovirus was known to be effective in gene therapy and easily delivered into a body owing to being produced in high titer and easily concentrated, clinical cancer gene therapy mediated by recombinant adenovirus has rapidly increased in the past five years. When cancer is treated using gene therapy mediated by recombinant adenovirus, since prolonged and continuous expression of therapeutic genes is not required, and host immune response induced by virus or viral proteins is not essential and can be advantageous in some cases, adenovirus become attractive as a gene transfer vehicle for cancer therapy (Pallard, F. Hum. Gene Ther., 9:283-286, 1998).
Most recombinant adenoviruses for cancer therapy, which carry a single therapeutic gene, are gene transfer systems first used in cancer gene therapy. Recently, efforts have been made to improve therapeutic efficacy of gene therapy through simultaneous expression of two therapeutic genes encoding proteins with different functions, rather than expression of one gene, or adenovirus-mediated gene therapy in combination with administration of antitumuor agents or radiotherapy, which have been commonly used for cancer therapy (Roth, J. A. et al., J. Nat. Cancer Ins., 89:21-39, 1997). For example, Korean Pat. No. 1997-5206 discloses a method of treating cancer using recombinant adenovirus harboring the p53 gene, known to have an antitumor effect, in which the recombinant adenovirus does not produce replication-competent viral particles and thus displays effective antitumor activity.
However, it has been reported that such replication-incompetent adenoviral vectors can induce antitumor activity in only primary infected cells or a very small number of surrounding cells. Therefore, a great number of recombinant adenovirus should be administered at once or administered repeatedly over several times, thus inducing cellular immunity and limiting its clinical applications. To overcome such problems, a variety of efforts have been made to develop a modified adenovirus capable of selectively replicating in and killing tumor cells, since the McCormick research group reported a recombinant adenovirus. The E1B 55 kDa gene-deleted adenovirus ONYX-015 (d11520), which was developed by McCormick, selectively replicates and induces cytolysis in tumor cells lacking functional p53 (Heise, C. et al., Nature Med., 3:639-645, 1997). In clinical trials for the treatment of head and neck cancer, the recombinant adenovirus ONYX-015 showed excellent therapeutic efficacy. Furthermore, another type recombinant adenovirus prepared by inserting a cancer-specific gene-regulatory region into E1 region was developed, in which viral proliferation is induced in a cancer tissue-specific manner. Also, antitumor effect and safety of a recombinant adenoviral vector can be improved by inserting the herpes simplex virus-thymidine kinase (HSV-TK) gene or CD gene into the E1 region, and thus the recombinant adenovirus loses its proliferation capacity (Freytag et al., Nat. Biotech., 15:866-870, 1997). In this regard, adenovirus-mediated gene therapy can be clinically applied under various circumstances. In particular, the recombinant adenovirus with tumor cell-specific cytotoxic effect was demonstrated to be more effective in cancers associated with mutations in the p53 gene, such as brain cancer, which is resistant to previous chemotherapies or radiation therapies (Shinoura, N. et al., Cancer Res. 59:3411-3416, 1999). However, administration of a high titer of adenovirus to brain is limited by its toxicity. Thus, methods of enhancing gene delivery efficiency should be developed in order to reduce the administration amount of adenovirus.
Adenovirus infects host cells mainly through coxsackievirus and adenovirus receptor (CAR) on the host cells (Tomko, R. P. et al., Proc. Natl. Sci. USA 94:3352-3356, 1997). Most cells in the host typically express sufficient amount of CAR, but no or little expression of CAR is found in muscle cells in matured bone tissues, lymphocytes, fibrocytes, pulmonary macrophages and some tumor cells, where adenovirus-mediated gene transfer efficiency is relatively low. Recent clinical trials using adenovirus as a gene delivery vehicle demonstrated that poor gene delivery into several tumor cells is attributed to lack of expressed CAR on the tumor cells. In addition, the recombinant adenovirus can infect normal cells with relatively high expression of CAR rather than target cells (tumor cells) with a low CAR level, resulting in reduction of its infection rate into tumor cells. To overcome such low transduction efficiency and lack of specificity for target cells by the recombinant adenovirus, there is a need for development of high-titer adenovirus. However, the high-titer adenoviral vectors can have increased toxicity and induce immune response in the host, thus threatening safe and effective clinical cancer therapy.
The disadvantages of recombinant adenoviral vectors in gene therapy owing to the above reasons can be overcome through infection of adenovirus into target cells in a CAR-independent manner. Effective gene transfer on epithermal cell was found when adenovirus type 2 fiber protein is replaced with that of adenovirus type 17. Also, a chimeric adenovirus prepared by replacing the knob domain of adenovirus type 5 with the knob domain of adenovirus subgroup B was demonstrated to effectively infect bone marrow cells not easily infected with adenovirus type 5. Wickham, T. J. J. reported that introduction of a polylysine motif or Arg-Gly-Asp (RGD)-containing peptide motif at the C-terminal region of adenovirus fiber protein allows adenovirus to specifically recognize alternative receptors, cell surface receptors including heparin and the integrin receptor, leading to successful infection of the virus (Wickham, T. J. J. Virol., 71:8221-8229, 1997). In addition, Kransnykh, V. et al., reported that transduction efficiency of adenovirus can be increased by inserting a targeting group capable of recognizing and then binding a target cell-specific receptor into the HI loop of the adenovirus fiber (Kransnykh, V. et al., J. Viro., 72:1884-1852, 1998; Yoshida, Y. et al., Hum. Gene. Ther. 9:2503-2515, 1998; and Shinoura, N. et al., Cancer Res. 59:3411-3416, 1999). However, such modifications were not sufficient for improvement of the transduction efficiency of adenovirus, and there are still efforts to develop a method of increasing the trasduction efficiency of adenovirus.
A plurality of patents and papers are referred and cited herein. All references cited herein are incorporated herein by reference in their entireties, and the current state of the conventional techniques in the art and the features of the present invention will be more clearly understood with the cited references.